EP4368640A1 - Anticorps cd19 et son application - Google Patents

Anticorps cd19 et son application Download PDF

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Publication number
EP4368640A1
EP4368640A1 EP22837042.5A EP22837042A EP4368640A1 EP 4368640 A1 EP4368640 A1 EP 4368640A1 EP 22837042 A EP22837042 A EP 22837042A EP 4368640 A1 EP4368640 A1 EP 4368640A1
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sequences
seq
antibody
identity
nos
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German (de)
English (en)
Inventor
Lian XIN
Qiong Wang
Cuiqing YANG
Zhuoxiao CAO
Renhong TANG
Jinsheng Ren
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Hainan Simcere Zaiming Pharmaceutical Co Ltd
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Hainan Simcere Zaiming Pharmaceutical Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present disclosure relates to the field of antibodies, and specifically to a CD19 antibody and an application thereof.
  • CD19 antigen belongs to the immunoglobulin superfamily and is a 95 kDa type I transmembrane glycoprotein, with a single transmembrane domain, a cytoplasmic C-terminus and an extracellular N-terminus.
  • the extracellular domain consists of two C2-type Ig-like domains, and the highly conserved cytoplasmic domain consists of 242 amino acids and 9 tyrosine residues near the C-terminus.
  • CD19 is critically involved in establishing intrinsic B cell signaling thresholds through modulating both B cell receptor-dependent and independent signaling.
  • CD19 interacts with other B cell receptors or surface molecules to directly or indirectly recruit and bind to various downstream protein kinases, including Src family (Lyn, Fyn), Ras family, Abl, Btk, adapter molecules (Vav, Grb2) and PI3K protein kinases.
  • Src family Lyn, Fyn
  • Ras family Ras family
  • Abl Abl
  • Btk adapter molecules
  • Vav, Grb2 adapter molecules
  • PI3K protein kinases PI3K protein kinases.
  • TAPA-1 tetraspanin CD81
  • CD225 functions as the dominant signaling component of a multi-molecular complex on the surface of mature B cells.
  • CD19 is expressed specifically in normal B cells and follicular dendritic cells, but not in pluripotent blood stem cells. During B cell lymphopoiesis, the surface expression of CD19 first takes place around the time of immunoglobulin gene rearrangement. The surface density of CD19 is highly regulated throughout B cell development and maturation, until the loss of expression during terminal plasma cell differentiation. CD19 is also expressed in most B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia and some acute myelogenous leukemia.
  • B cells provide humoral immune components of the adaptive immune system by secreting antibodies, interacting with T cells and producing chemokines and cytokines, thus playing a key role in host defense.
  • B cell subsets particularly plasma cells and plasmablasts, have long been considered as the main source in antibody production.
  • B cells are involved in the pathogenesis of many autoimmune diseases, including central nervous system (CNS) diseases, multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD). Deficiency of CD19 in experimental animals and humans can lead to impaired humoral responses, resulting in increased susceptibility to infection. However, overexpression of human CD19 can lead to autoimmune diseases in tight skin (TSK/+) mice (model of human systemic sclerosis (SSc)).
  • TSK/+ tight skin
  • SSc model of human systemic sclerosis
  • CD19 can be used as a landmark target for the treatment of B cell related leukemia and autoimmune diseases. Antibodies having good affinity to human CD19 are needed.
  • the present disclosure provides an antibody or antigen binding fragment specifically binding to human CD19, a multi-characteristic antigen binding molecule, a chimeric antigen receptor, an immune effector cell, a nucleic acid molecule, a vector, a cell, a preparation method, a pharmaceutical composition, a pharmaceutical use, and a disease treatment method, wherein the antibody or antigen binding fragment has good binding ability to human CD 19, improving human CD19-related detection, treatment or other applications related to human CD19 binding.
  • the present disclosure discloses an antibody or antigen binding fragment specifically binding to human CD19, the antibody or antigen binding fragment comprises a heavy chain variable region and/or a light chain variable region, the heavy chain variable region comprises HCDR1-3, the light chain variable region comprises LCDR1-3,
  • the HCDR1-3 comprise sequences as shown in SEQ ID NOs: 115-117, 121-123, 127-129, 133-135, 139-141, 145-147, 151-153, 157-159, 163-165, 169-171, 175-177, 181-183, 187-189, 193-195, 199-201, 205-207, 211-213, 217-219, 223-225, 229-231, 235-237, 241-243, 247-249, 253-255, 259-261, 265-267, 271-273, 277-279, 283-285, 289-291, 295-297, 301-303, 307-309, 313-315, 319-321, 325-327, 331-333, 337-339, 343-345, 349-351, 355-357, 361-363, 367-369, 373-375, 379-381, 385-387, 391-393, 397-399, 403-405, 409-411
  • the antibody or antigen binding fragment comprises the heavy chain variable region and the light chain variable region
  • the HCDR1-3 and the LCDR1-3 comprise sequences selected from the group consisting of:
  • the heavy chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 439-442, or a sequence having at least 70% identity or at most 15 amino acid differences compared thereto; and the light chain variable region comprises a sequence as shown in any one of SEQ ID NOs: 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
  • the at least 70% identity is preferably at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity;
  • the at most 3 mutations are preferably at most 3, 2, 1 or 0 mutations;
  • the at most 15 mutations are preferably at most 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 mutations; and preferably, the mutation is an insertion, a deletion or a substitution, the substitution is preferably a conservative amino acid substitution, and the mutation is preferably a back mutation or a hotspot mutation.
  • the antibody or antigen binding fragment further comprises a heavy chain constant region and/or a light chain constant region;
  • the antibody or antigen binding fragment is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a mono-specific antibody, a multi-specific antibody (for example, a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, a fully human antibody, Fab, Fab', Fab'-SH, F(ab')2, Fd, Fv, scFv, a diabody or a single domain antibody.
  • the antibody or antigen binding fragment further comprise a conjugate; and the conjugate can be selected from a therapeutic agent or a tracer, the therapeutic agent can be selected from a radioisotope, a chemotherapeutic drug or an immunomodulator, and the tracer can be selected from a radiological contrast agent, a paramagnetic ion, a metal, a fluorescent tag, a chemiluminescence tag, an ultrasonic contrast agent and a photosensitizer.
  • the antibody or antigen binding fragment binds to human CD19 with a KD value of less than 1E-08M, 1E-09M, 1E-10M or 1E-11M; preferably, the antibody or antigen binding fragment also binds to monkey CD19, more preferably, the antibody or antigen binding fragment binds to monkey CD19 with a KD value of less than 1E-8M, 1E-9M, 1E-10M, 1E-11M or 1E-12M.
  • the present disclosure further discloses a multi-specific antigen binding molecule, wherein the multi-specific antigen binding molecule comprises at least a first antigen binding module and a second antigen binding module, the first antigen binding module comprises the aforementioned antibody or antigen binding fragment, and the second antigen binding module binds to other targets different from the first antigen binding module or binds to different epitopes of the same target; preferably, the second antigen binding module is an antibody or an antigen binding fragment; and preferably, the other targets are selected from the group consisting of: (1) a tumor specific antigen (TSA) or a tumor associated antigen (TAA); (2) an immune checkpoint; and (3) a target that recruits and/or activates immune cells.
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • the present disclosure further discloses a chimeric antigen receptor (CAR), wherein the chimeric antigen receptor comprises at least an extracellular antigen binding domain, a transmembrane domain and an intracellular signaling domain, and the extracellular antigen binding domain comprises the aforementioned antibody or antigen binding fragment or the aforementioned multi-specific antigen binding molecule.
  • CAR chimeric antigen receptor
  • the present disclosure further discloses an immune effector cell, wherein the immune effector cell expresses the aforementioned chimeric antigen receptor and/or comprises a nucleic acid molecule encoding the aforementioned chimeric antigen receptor;
  • the present disclosure further discloses an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes the aforementioned antibody or antigen binding fragment, the aforementioned multi-specific antigen binding molecule or the aforementioned chimeric antigen receptor.
  • the present disclosure further discloses a vector, wherein the vector comprises the aforementioned nucleic acid molecule.
  • the present disclosure further discloses a cell, wherein the cell comprises the aforementioned vector.
  • the present disclosure further discloses a method for preparing the aforementioned antibody or antigen binding fragment or the aforementioned multi-specific antigen binding molecule, wherein the method comprises: (1) culturing the aforementioned cell, and/or (2) isolating the antibody or antigen binding fragment, or the multi-specific antigen binding molecule expressed by the cell.
  • the present disclosure further discloses a method for preparing the aforementioned immune effector cell, wherein the method comprises: introducing a nucleic acid molecule encoding the aforementioned chimeric antigen receptor into the immune effector cell, and/or initiating the immune effector cell to express the chimeric antigen receptor.
  • the present disclosure further discloses a pharmaceutical composition, wherein the pharmaceutical composition comprises the aforementioned antibody or antigen binding molecule, the aforementioned multi-specific antigen binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell or a product prepared according to the aforementioned method; preferably, the composition further comprises a pharmaceutically acceptable carrier, diluent or auxiliary agent.
  • the present disclosure further discloses the use of the aforementioned antibody or antigen binding fragment, the aforementioned multi-specific antigen binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition, or a product prepared according to the aforementioned method in the preparation of a drug for treating a CD19-related disease; preferably, the CD19-related disease is selected from or an autoimmune disease; and more preferably, the tumor is selected from lymphoma or leukemia, such as B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma or multiple myeloma, and the autoimmune disease is selected from an autoimmune disease of the nervous system, a rheumatoid disease, systemic lup
  • the present disclosure further discloses a method for treating a CD19-related disease, wherein the method comprises administering to a subject an effective amount of a drug, and the drug comprises the aforementioned antibody or antigen binding fragment, the aforementioned multi-specific antigen binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition, or a product prepared according to the aforementioned method, preferably, the CD19-related disease is selected from a tumor or an autoimmune disease; and more preferably, the tumor is selected from lymphoma or leukemia, such as B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma or multiple myeloma, and the autoimmune disease is selected from an autoimmune disease of the nervous system
  • the present disclosure further discloses the aforementioned antibody or antigen binding fragment, the aforementioned multi-specific antigen binding molecule, the aforementioned immune effector cell, the aforementioned nucleic acid molecule, the aforementioned vector, the aforementioned cell, the aforementioned pharmaceutical composition, or a product prepared according to the aforementioned method, for use in a drug for treating a CD19-related disease
  • the CD19-related disease is selected from a tumor or an autoimmune disease
  • the tumor is selected from lymphoma or leukemia, such as B cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, non-Hodgkin lymphoma, mantle cell lymphoma, follicular lymphoma, diffuse large B cell lymphoma or multiple myeloma
  • the autoimmune disease is selected from an autoimmune disease of the nervous system, a rheumatoid disease, systemic
  • the CD 19 antibody and the antigen binding fragment thereof of the present disclosure have good binding ability to human CD19, wherein some of the antibodies can also cross-bind to monkey CD19, thereby providing great significance for the development of CD19-related antibody products or cell therapy products and has good application prospects.
  • compositions comprising A and B
  • composition containing other components in addition to A and B fall within the scope of the aforementioned "a composition”.
  • an antigen binding molecule typically specifically binds to an antigen and substantially the same antigen with a high affinity, but does not bind to an unrelated antigen with a high affinity. Affinity is generally reflected by the equilibrium dissociation constant (KD), where lower KD indicates higher affinity.
  • KD equilibrium dissociation constant
  • a high affinity typically means having a KD of about 10 -7 M or less, about 10 -8 M or less, about 1 ⁇ 10 -9 M or less, about 1 ⁇ 10 -10 M or less, about 1 ⁇ 10 -11 M or less, or about 1 ⁇ 10 -12 M or less.
  • KD Kd/Ka, where Kd represents the dissociation rate and Ka represents the binding rate.
  • the equilibrium dissociation constant KD can be measured using well-known methods in the art, such as surface plasmon resonance (e.g., Biacore) or equilibrium dialysis assay. Exemplarily, see the method for obtaining the KD value as shown in example 8 herein.
  • antigen binding molecule is used herein in the broadest sense and refers to a molecule specifically binding to an antigen.
  • the antigen binding molecule includes, but is not limited to, an antibody or an antibody mimetic.
  • the "antibody mimetic” refers to an organic compound or a binding domain that can specifically bind to an antigen, but is not structurally related to an antibody.
  • the antibody mimetic includes, but is not limited to, affibody, affitin, affilin, a designed ankyrin repeat protein (DARPin), a nucleic acid aptamer or a Kunitz-type domain peptide.
  • DARPin ankyrin repeat protein
  • antibody is used herein in the broadest sense and refers to a polypeptide or a combination of polypeptides comprising sufficient sequences from a heavy chain variable region of an immunoglobulin and/or sufficient sequences from a light chain variable region of an immunoglobulin to be able to specifically bind to an antigen.
  • the “antibody” herein encompasses various forms and various structures as long as they exhibit the desired antigen-binding activity.
  • the “antibody” herein includes an alternative protein scaffold or artificial scaffold with grafted complementarity determining regions (CDRs) or CDR derivatives.
  • Such scaffolds include antibody-derived scaffolds comprising mutations introduced, e.g., to stabilize the three-dimensional structure of the antibody, and fully synthetic scaffolds comprising, e.g., biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1): 121-129 (2003 ); Roque et al., Biotechnol. Prog. 20: 639-654 (2004 ).
  • Such scaffolds may also include non-antibody-derived scaffolds, for example, scaffold proteins known in the art which can be used for grafting CDRs, including but not limited to tenascin, fibronectin, a peptide aptamer, etc.
  • the “antibody” herein includes a typical "four-chain antibody”, which belongs to an immunoglobulin consisting of two heavy chains (HC) and two light chains (LC);
  • the heavy chain refers to a polypeptide chain that consists of, in a direction from the N-terminus to the C-terminus, a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; moreover, when the full-length antibody is an IgE isotype, it optionally also comprises a heavy chain constant region CH4 domain;
  • the light chain is a polypeptide chain consisting of, in a direction from the N-terminus to the C-terminus, a light chain variable region (VL) and a light chain constant region (CL); the heavy chain is linked to the heavy chain and the heavy chain is linked to the light chain via disulfide bonds, forming a Y-shaped structure.
  • immunoglobulin can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and the corresponding heavy chains thereof are a ⁇ chain, a ⁇ chain, a ⁇ chain, an ⁇ chain and an ⁇ chain, respectively.
  • the same class of Ig can also be divided into different subclasses according to the differences in the amino acid composition of the hinge region thereof and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3 and IgG4, and IgA can be divided into IgA1 and IgA2.
  • the light chains are divided into a ⁇ chain or a ⁇ chain by the difference in the constant region.
  • Each Ig class of the five Ig classes can have either a ⁇ chain or a ⁇ chain.
  • the “antibody” herein also includes an antibody that does not comprise light chains, for example, heavy-chain antibodies (HCAbs) produced from Camelus dromedarius, Camelus bactrianus, Lama glama, Lama guanicoe and Vicugna pacos, and Ig new antigen receptors (IgNARs) found in Chondrichthyes such as sharks.
  • HCAbs heavy-chain antibodies
  • IgNARs Ig new antigen receptors
  • the "antibody” herein may be derived from any animal, including but not limited to humans and non-human animals selected from primates, mammals, rodents and vertebrates, such as Camelidae, Lama glama, Lama guanicoe, Vicugna pacos, sheep, rabbits, mice, rats or Chondrichthyes (such as sharks).
  • the "antibody” herein includes but is not limited to a monoclonal antibody, a polyclonal antibody, a mono-specific antibody, a multi-specific antibody (for example, a bispecific antibody), a monovalent antibody, a multivalent antibody, an intact antibody, a fragment of an intact antibody, a naked antibody, a conjugated antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.
  • the term "monoclonal antibody” herein refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, individual antibodies comprising the population are identical and/or bind to the same epitope, except for possible variants (such as those containing naturally occurring mutations or produced during the production of a preparation, such variants generally being present in a small amount).
  • each monoclonal antibody in monoclonal antibody preparations targets a single determinant on an antigen.
  • the modifier "monoclonal” herein should not be construed as requiring the production of the antibody or antigen binding molecule by any particular method.
  • monoclonal antibodies can be prepared by a variety of techniques, including (but not limited to) hybridoma techniques, recombinant DNA methods, phage library display techniques, methods using transgenic animals containing all or part of human immunoglobulin loci, and other methods known in the art.
  • natural antibody herein refers to an antibody produced and paired by the immune system of a multicellular organism.
  • the antibody of the term “engineered antibody” herein refers to a non-natural antibody obtained by techniques such as genetic engineering and antibody engineering.
  • the "engineered antibody” includes a humanized antibody, a small molecule antibody (such as scFv), a bispecific antibody, etc.
  • the term "monospecific” herein refers to having one or more binding sites, wherein each binding site binds to the same epitope of the same antigen.
  • multi-specific antibody herein refers to an antibody having at least two antigen binding sites, each of which binds to a different epitope of the same antigen or to a different epitope of a different antigen. Therefore, terms such as “bispecific”, “trispecific” and “tetraspecific” refer to the number of different epitopes to which an antibody/antigen binding molecule can bind.
  • valence herein refers to the presence of a defined number of binding sites in an antibody/an antigen binding molecule. Therefore, the terms “monovalent”, “bivalent”, “tetravalent” and “hexavalent” indicate the presence of one binding site, two binding sites, four binding sites and six binding sites in an antibody/antigen binding molecule, respectively.
  • full-length antibody “complete antibody” and “intact antibody” are used interchangeably herein and mean that they have a structure substantially similar to that of a natural antibody.
  • the "antigen binding fragment” and “antibody fragment” are used interchangeably herein, which do not possess the full structure of an intact antibody, and only comprise a part of an intact antibody or a variant of the part, wherein the part of the intact antibody or the variant of the part has the ability to bind to an antigen.
  • the "antigen binding fragment” or “antibody fragment” herein includes but is not limited to Fab, Fab', Fab'-SH, F(ab') 2 , Fd, Fv, scFv, a diabody and a single domain antibody.
  • Fab fragments each containing a variable domain of heavy chain and a variable domain of light chain, and also a constant domain of the light chain and the first constant domain (CH1) of the heavy chain.
  • Fab fragment refers to an antibody fragment comprising the VL domain and the constant domain (CL) of a light chain, and the VH domain and the first constant domain (CH1) of a heavy chain.
  • the Fab' fragment differs from the Fab fragment by adding a small number of residues at the carboxyl terminus of the CH1 domain of the heavy chain, including one or more cysteines from the hinge region of the antibody.
  • Fab'-SH is a Fab' fragment in which the cysteine residue of the constant domain carries a free thiol group. Treatment with pepsin produces a F(ab') 2 fragment having two antigen binding sites (two Fab fragments) and a portion of the Fc region.
  • Fd refers to an antibody consisting of VH and CH1 domains.
  • Fv refers to an antibody fragment consisting of a single arm VL and a VH domain.
  • the Fv fragment is generally considered to be the smallest antibody fragment that can form an intact antigen binding site. It is generally believed that the six CDRs confer antigen binding specificity to an antibody. However, even one variable region (such as a Fd fragment, which contains only three antigen-specific CDRs) is able to recognize and bind to an antigen, although it may have a lower affinity than an intact binding site.
  • scFv single-chain variable fragment
  • scFv single-chain variable fragment
  • a linker see, for example, Bird et al., Science 242: 423-426 (1988 ); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988 ); and Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Roseburg and Moore eds., Springer-Verlag, New York, pages 269-315 (1994 )).
  • Such scFv molecules can have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable linkers in the prior art consist of repeated GGGGS (SEQ ID NO.450) amino acid sequences or variants thereof.
  • linkers having an amino acid sequence (GGGGS) 4 (SEQ ID NO.451) can be used, but variants thereof can also be used ( Holliger et al., (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448 ).
  • Other linkers that can be used in the present disclosure are described by Alfthan et al., (1995), Protein Eng.
  • a disulfide bond can also be present between VH and VL of scFv, forming a disulfide-linked Fv (dsFv).
  • VH and VL domains of the term "diabody” herein are expressed on a single polypeptide chain, but a linker used is too short to allow for pairing between two domains of the same chain, thereby forcing the domain to pair with the complementary domain of the other chain and producing two antigen binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993 ), and Poljak R.J. et al., Structure 2: 1121-1123 (1994 )).
  • single domain antibody Single domain antibody (sdAb)
  • VHH single domain antibody
  • nanobody single domain antibody consisting of only one heavy chain variable region constructed by cloning the heavy chain variable region of an antibody, which is the smallest antigen binding fragment with full functionality.
  • a single domain antibody consisting of only one heavy chain variable region is constructed by obtaining an antibody that naturally lacks light chains and heavy chain constant region 1 (CH1), and then cloning the heavy chain variable region of the antibody.
  • Single domain antibody can be derived from a Camelidae heavy chain antibody or Chondrichthyes fish IgNAR.
  • naked antibody herein refers to an antibody that is not conjugated with a therapeutic agent or a tracer
  • conjugated antibody herein refers to an antibody that is conjugated with a therapeutic agent or a tracer
  • chimeric antibody herein refers to an antibody in which a portion of the light chain or/and heavy chain thereof is derived from one antibody (which may be derived from a particular species or belong to a particular antibody class or subclass), and another portion of the light chain or/and the heavy chain is derived from another antibody (which may be derived from the same or a different species or belong to the same or a different antibody class or subclass), but which in any case retains the activity of binding to an antigen of interest ( U.S.P 4,816,567, to Cabilly et al .; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984 )).
  • chimeric antibody may include an antibody (e.g., a human-murine chimeric antibody) in which the heavy and light chain variable regions of the antibody are derived from a first antibody (e.g., a murine-derived antibody) and the heavy and light chain constant regions of the antibody are derived from a second antibody (e.g., a human antibody).
  • a first antibody e.g., a murine-derived antibody
  • a second antibody e.g., a human antibody
  • humanized antibody herein refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to increase sequence homology with a human antibody.
  • all or part of the CDR regions of a humanized antibody come from a non-human antibody (a donor antibody), and all or part of the non-CDR regions (for example, a variable region FR and/or a constant region) come from a human immunoglobulin (a receptor antibody).
  • a humanized antibody generally retains or partially retains the expected properties of a donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to improve immune cell activities, ability to enhance immune responses, etc.
  • the term "fully human antibody” herein refers to an antibody having variable regions in which both the FRs and CDRs are derived from human germline immunoglobulin sequences. Furthermore, if the antibody comprises a constant region, the constant region also is derived from human germline immunoglobulin sequences.
  • the "fully human antibody” herein may include amino acid residues not encoded by human germline immunoglobulin sequences (for example, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the "fully human antibody” herein does not include an antibody in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences.
  • variable region herein refers to a region in the heavy or light chain of an antibody that is involved in enabling the antibody to bind to an antigen.
  • VH Heavy chain variable region
  • VL light chain variable region
  • LCVR light chain variable region
  • the variable domains of the heavy and light chains (VH and VL, respectively) of a natural antibody generally have similar structures, and each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p.91 (2007 ).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • CDR complementarity determining region
  • HVR hypervariable region
  • VH heavy chain variable region
  • VL light chain variable region
  • LCDR complementarity determining region
  • framework region or “FR region” is interchangeable and refers to those amino acid residues other than CDRs in the heavy chain variable region or light chain variable region of an antibody.
  • a typical antibody variable region consists of 4 FR regions and 3 CDR regions in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • CDR can be defined by the Kabat numbering system. Tool websites include abYsis website (www.abysis.org/abysis/sequence input/key_ annotation/key_annotation.cgi)
  • Kabat numbering system herein generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Md., 1991 ).
  • the term “heavy chain constant region” herein refers to the carboxyl terminus portion of an antibody heavy chain, which is not directly involved in the binding of an antibody to an antigen, but exhibits an effector function, such as an interaction with an Fc receptor, and has a more conservative amino acid sequence relative to the variable domain of an antibody.
  • the “heavy chain constant region” comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or variants or fragments thereof.
  • the “heavy chain constant region” includes a "full-length heavy chain constant region” and a “heavy chain constant region fragment", the former has a structure substantially similar to that of a natural antibody constant region, while the latter only includes “a portion of the full-length heavy chain constant region”.
  • a typical "full-length antibody heavy chain constant region” consists of CH1 domain-hinge region-CH2 domain-CH3 domain, which also includes a CH4 domain when an IgE antibody is referred to, and does not include the CH1 domain when a heavy chain antibody is referred to.
  • a typical "heavy chain constant region fragment” can be selected from CH1, Fc or CH3 domains.
  • light chain constant region refers to the carboxyl terminus portion of an antibody light chain, which is not directly involved in the binding of an antibody to an antigen, and the light chain constant region can be selected from a constant ⁇ domain or a constant ⁇ domain.
  • Fc refers to the carboxyl terminus portion of an intact antibody obtained by hydrolyzing the antibody with papain, which typically comprises CH3 and CH2 domains of an antibody.
  • the Fc region includes, for example, an Fc region of a natural sequence, a recombinant Fc region and a variant Fc region.
  • the boundary of the Fc region of an immunoglobulin heavy chain can be slightly changed, the Fc region of a human IgG heavy chain is usually defined as the region extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus.
  • the C-terminal lysine of the Fc region (residue 447 according to the Kabat numbering system) can be removed, for example, during the production or purification of an antibody, or by recombinant engineering of a nucleic acid encoding an antibody heavy chain, so the Fc region may or may not include Lys447.
  • conservative amino acid generally refers to amino acids that belong to the same class or have similar characteristics (such as charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation and rigidity).
  • conservative amino acid residues a substitution of amino acid residues in the group is a substitution of conservative amino acids:
  • identity herein can be calculated by the following method: to determine the percent "identity" of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (for example, gaps can be introduced in either or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When the position in the first sequence is occupied by the same amino acid residue or nucleotide as that at the corresponding position in the second sequence, the molecules are identical at that position.
  • the percent identity between two sequences will vary with the identical positions shared by the sequences, considering the number of gaps that need to be introduced to optimally align the two sequences and the length of each gap.
  • sequence comparison and the calculation of percent identity between two sequences can be achieved using a mathematical algorithm.
  • percent identity between two amino acid sequences is determined using the Needlema and Wunsch algorithm ((1970) J. Mol. Biol. 48: 444-453 ) in the GAP program that has been integrated into the GCG software package (available at www.gcg.com) and using the Blossum 62 matrix or the PAM250 matrix, with a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com) and using the NWSgapdna.CMP matrix, with a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • a particularly preferred parameter set is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a gap frameshift penalty of 5.
  • the percent identity between two amino acid sequences or nucleotide sequences can also be determined using the PAM120 weighted remainder table, with a gap length penalty of 12 and a gap penalty of 4, and using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17) that has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid and protein sequences of the present disclosure can further be used as "query sequences" to perform searches against public databases, e.g., to identify other family member sequences or related sequences.
  • the NBLAST and XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215: 403-10 can be used to perform such searches.
  • gapped BLAST can be used as described in Altschul et al., (1997) Nucleic Acids Res. 25: 3389-3402 .
  • the default parameters of the corresponding programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST the default parameters of the corresponding programs. See www.ncbi.nlm.nih.gov.
  • chimeric antigen receptor herein refers to an artificial cell surface receptor engineered to express on immune effector cells and specifically bind to an antigen, comprising at least (1) an extracellular antigen binding domain, such as a variable heavy chain or light chain of an antibody, (2) a transmembrane domain anchoring the CAR into an immune effector cell, and (3) an intracellular signaling domain.
  • the CAR can redirect T cells and other immune effector cells to selected targets, such as cancer cells, in a non-MHC-restricted way by using an extracellular antigen-binding domain.
  • nucleic acid herein includes any compound and/or substance comprising a polymer of nucleotides.
  • Each nucleotide consists of a base, specifically a purine or pyrimidine base (i.e., cytosine (C), guanine (G), adenine (A), thymine (T) or uracil (U)), a sugar (i.e., deoxyribose or ribose) and a phosphate group.
  • cytosine C
  • G guanine
  • A adenine
  • T thymine
  • U uracil
  • a nucleic acid molecule is described by a sequence of bases, whereby the bases represent the primary structure (linear structure) of the nucleic acid molecule.
  • the sequence of bases is usually expressed as 5' to 3'.
  • nucleic acid molecule encompasses deoxyribonucleic acid (DNA), including for example complementary DNA (cDNA) and genomic DNA, ribonucleic acid (RNA), especially messenger RNA (mRNA), synthetic forms of DNA or RNA, and a polymer comprising a mixture of two or more of these molecules.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • mRNA messenger RNA
  • the nucleic acid molecule can be linear or circular.
  • nucleic acid molecule includes both sense strand and antisense strand, as well as single-stranded form and double-stranded form.
  • nucleic acid molecules described herein may contain naturally occurring or non-naturally occurring nucleotides.
  • nucleic acid molecule also encompasses DNA and RNA molecules which are suitable as vectors for direct expression of the antibody of the present disclosure in vitro and/or in vivo, for example in a host or patient.
  • DNA e.g., cDNA
  • RNA e.g., mRNA
  • mRNA can be chemically modified to enhance the stability of the RNA vector and/or the expression of the encoded molecule, so that the mRNA can be injected into a subject to generate an antibody in vivo (see, for example, Stadler et al., Nature Medicine 2017, Published online June 12, 2017, doi: 10.1038/nm.4356 or EP 2 101 823 B1 ).
  • isolated nucleic acid herein refers to a nucleic acid molecule that has been separated from components of the natural environment thereof.
  • the isolated nucleic acid includes a nucleic acid molecule contained in a cell that normally contains the nucleic acid molecule, but which is present extrachromosomally or at a chromosomal location other than the natural chromosomal location thereof.
  • vector refers to a nucleic acid molecule capable of amplifying another nucleic acid to which it is linked.
  • the term includes a vector having a self-replicating nucleic acid structure and a vector that is integrated into the genome of a host cell into which the vector has been introduced.
  • Certain vectors can direct the expression of nucleic acid to which they are operably linked, and such vectors are referred to herein as "expression vectors”.
  • the term "host cell” herein refers to a cell into which a foreign nucleic acid is introduced, including the progenies of such a cell.
  • the host cell includes "transformant” and “transformed cell” which include the primary transformed cell and progeny derived therefrom, regardless of the number of passages.
  • the progeny may not be identical to the parental cell in nucleic acid content, and may contain a mutation.
  • the mutant progeny with the same function or biological activity as those screened or selected in the initially transformed cells are included herein.
  • the term "pharmaceutical composition” refers to a preparation that is present in a form which allows the active ingredients contained therein to be biologically effective and does not contain additional ingredients that would be unacceptably toxic to the subject to which the pharmaceutical composition is administered.
  • the pharmaceutical composition described herein can comprise a plurality of active ingredients.
  • the pharmaceutical composition can further comprise other active ingredients, such as a chemotherapeutic drug or an immune checkpoint inhibitor.
  • treatment refers to surgical or therapeutic treatment, the purpose of which is to prevent and slow down (reduce) an undesired physiological change or pathology, such as cancer progression, in a subject being treated.
  • Beneficial or desired clinical outcomes include, but are not limited to, the alleviation of symptoms, the weakening of disease severity, the stabilization of disease state (i.e., no deterioration), the delay or slowing down of disease progression, the amelioration or mitigation of disease state, and remission (whether partial or complete), whether detectable or undetectable.
  • Objects in need of treatment include those who already have a disorder or disease, those who are susceptible to a disorder or disease or those who intend to prevent a disorder or disease.
  • slowing down, alleviation, weakening, mitigation, and remission are referred to, their meanings also include elimination, disappearance, non-occurrence and other circumstances.
  • subject refers to an organism receiving treatment for a particular disease or disorder as described in the present disclosure.
  • subjects and patients include mammals, such as humans, primates (e.g., monkeys) or non-primate mammals, receiving treatment for a disease or a disorder.
  • a therapeutically effective dose refers to an amount of a therapeutic agent effective to prevent or relieve a disease or a disorder or the progression of the disease when administered alone or in combination with another therapeutic agent to a cell, tissue or subject.
  • the “effective amount” also refers to an amount of a compound sufficient to relieve symptoms, such as treating, curing, preventing or relieving related medical disorders, or increasing the speed of treating, curing, preventing or relieving these disorders.
  • a therapeutically effective dose refers to the ingredient alone.
  • a therapeutically effective dose refers to the combined dosage of the active ingredients that produce a therapeutic effect, whether administered in combination, sequentially or simultaneously.
  • cancer refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Both benign and malignant cancers are included in this definition.
  • tumor or “neoplasm” herein refers to all neoplastic cell growth and proliferation, whether malignant or benign, and to all pre-cancerous and cancerous cells and tissues.
  • cancer and “tumor” are not mutually exclusive when referred to herein.
  • EC50 refers to the half-maximal effective concentration, which includes the concentration of an antibody that induces a response halfway between baseline and maximum after a specified exposure time. EC50 essentially represents the concentration of an antibody at which 50% of the maximal effect thereof is observed and which can be measured by methods known in the art.
  • a His-tagged nucleotide sequence that contains a nucleotide sequence encoding the amino acid of human CD19 exon1-3 was cloned into pTT5 vector by General Biosystems (Anhui) Co., Ltd., and the plasmid was prepared according to the established standard molecular biological method. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press ).
  • HEK293E cells purchased from Suzhou Yiyan Biotechnology Co., Ltd.
  • PI Polysciences, catalog number: 24765-1
  • FreeStyle TM 293 Thermofisher scientific, catalog number: 12338018
  • Thermofisher scientific, catalog number: 12338018 was used for scale-up culture at 37°C.
  • the cell culture fluid was collected, centrifuged to remove cell components, and the culture supernatant containing human CD19 exon1-3 was obtained.
  • the culture supernatant was loaded onto a nickel ion affinity chromatography column HisTrap TM Excel (GE Healthcare, catalog number: GE17-3712-06), and the change in the ultraviolet absorbance (A280 nm) was monitored with an ultraviolet (UV) detector.
  • UV ultraviolet
  • the nickel ion affinity chromatography column was washed with 20 mM PB and 0.5 M NaCl (pH 7.4) until the ultraviolet absorbance returned to the baseline, and then gradient elutions (2%, 4%, 8%, 16%, 50% and 100%) were performed with buffer A: 20 mM PB and 0.5 M NaCl (pH 7.4), and buffer B: 20 mM PB, 0.5 M NaCl and 500 mM imidazole.
  • the His-tagged human CD19 exon1-3 protein eluted from the nickel ion affinity chromatography column was collected and dialyzed with a PBS phosphate buffer (pH 7.4) overnight at 4°C.
  • the dialyzed protein was aseptically filtered through a 0.22 ⁇ m filter membrane and then subpackaged for storage at -80°C to obtain a purified human CD19 exon1-3 protein.
  • the bands of interest of samples detected by SDS-PAGE on reducing and non-reducing gels were as shown in FIG. 1 .
  • FMC63 and 9G8 clones are antibodies that recognize human CD19, and the antigen binding epitopes of both are located in the membrane-proximal end.
  • the heavy chain variable region and light chain variable region sequences of FMC63 clone were obtained according to patent WO 2016033570 A1
  • the heavy chain variable region and light chain variable region sequences of 9G8 clone were obtained according to patent WO 2018083535 A1 .
  • the plasmids were prepared according to the established standard molecular biological method. For the specific method, see Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press ).
  • the expression vector was transiently transfected into HEK293E cells (purchased from Suzhou Yiyan Biotechnology Co., Ltd.) according to the instructions of PEI (purchased from Polysciences, catalog number: 24765-1), the transfected cells were continuously cultured at 37°C for 5 days using FreeStyle TM 293 (Thermofisher scientific, catalog number: 12338018), and the cell components were removed by centrifugation to obtain the culture supernatant containing the antibody.
  • the culture supernatant was loaded onto a protein A chromatography column (the protein A packing AT Protein A Diamond and the chromatography column BXK16/26 were both purchased from Bestchrom (Shanghai) Biosciences Ltd., catalog numbers: AA0273 and B-1620), and the column was washed with a PBS phosphate buffer (pH 7.4), then washed with 20 mM PB and 1 M NaCl (pH 7.2), and finally eluted with a citric acid buffer (pH 3.4).
  • a nucleotide sequence encoding the full-length amino acid sequence of human CD19 was cloned into vector pcDNA3.1 and a plasmid was prepared by General Biosystems (Anhui) Co., Ltd. Plasmid transfection (Lipofectamine ® 3000 Transfection Kit, purchased from Invitrogen, catalog number: L3000-015) was performed on CHO-K1 cell line (purchased from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and then the transfected cells were selectively incubated for 2 weeks in DMEM/F12 medium containing 10 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum.
  • FMC63-mIgG1 antibody as primary antibody and Alexa Fluor 647-labeled secondary antibody were used to sort positive monoclonal cells into a 96-well plate on flow cytometer FACS AriaII (BD Biosciences), and the plate was placed in a cell incubator at 37°C and 5% (v/v) CO 2 for cell culture. After about 2 weeks, some wells containing monoclonal cells were selected for expansion. The expanded clones were screened by flow cytometry. The monoclonal cell line with better growth and higher fluorescence intensity was selected for further scale-up culture and then cryopreserved in liquid nitrogen.
  • CHO-K1 monoclonal cell lines with high level of human CD19 expression have been prepared: CHO-K1-human CD19-2C8, CHO-K1-human CD19-1C4, CHO-K1-human CD19-2G4 and CHO-K1-human CD19 1C9.
  • the clone CHO-K1-human CD19-2C8 was selected for the subsequent experiment, and unless particularly stated, the CHO-K1-human CD19 cells used in the subsequent examples were all CHO-K1-human CD19-2C8.
  • a nucleotide sequence encoding the full-length amino acid sequence of monkey CD19 (NCBI: XM_005591542.1) was cloned into pcDNA3.1 vector (purchased from Thermofisher scientific) and a plasmid was prepared.
  • the transfected cells were selectively cultured in DMEM medium containing 10 ⁇ g/ml puromycin and 10% (v/v) fetal bovine serum for 2 weeks, subcloned in a 96-well culture plate by limited dilution method, and cultured in an incubator at 37°C and 5% (v/v) CO 2 . After about 2 weeks, some wells containing polyclonal cells were selected and the cells were expanded into a 6-well plate.
  • the expanded clones were screened by FACS with CD19 antibody 9G8-mIgG1 with monkey cross-activity as primary antibody and Alexa Fluor 647-labeled secondary antibody (Jackson, catalog number: 115-605-003), and the cell line with better growth and higher fluorescence intensity was selected for further scale-up culture and then cryopreserved in liquid nitrogen.
  • the results are as shown in Table 4 and FIG. 4 , indicating that the cell line that shows a positive cell peak overexpressing monkey CD19 has been screened and can be used to detect the cross-activity of antibodies.
  • Table 4 Results of HEK293T cell line stably transfected with monkey CD19 protein detected by FACS No. Clone number of stably transfected cell line Mean fluorescence intensity of cells IgG subtype control CD19 antibody 1 HEK293T-monkey CD19 62 66503
  • mice An anti-human CD19 monoclonal antibody was produced by immunizing mice.
  • BALB/c AnNCrl mice purchasedd from Beijing Vital River Laboratory Animal Technology Co., Ltd.
  • SJL/JorllcoCrl mice purchased from Shanghai SLAC Co., Ltd.
  • Female, aged 6-8 weeks were used in the experiment.
  • Breeding environment SPF rating. After the mice were purchased, they were raised in a laboratory environment for 1 week, with a 12/12-hour light/dark cycle adjustment, and a temperature of 20°C-25°C; humidity 40%-60%.
  • the acclimatized mice were immunized according to the following scheme.
  • the immunizing antigen was human CD19 (aa20-291)-huFc protein (purchased from ACRO Biosystems, catalog number: CD9-H5251) or CHO-K1 cell line stably transfected with human CD19 protein.
  • the immunogen was emulsified with TiterMax (purchased from Sigma, catalog number: T2684) and injected subcutaneously (SC) and intraperitoneally (IP) at 0.1 ml respectively, that is, each mouse was injected with 50 ⁇ g of the immunogen; and for booster immunization, the immunogen was injected subcutaneously at multiple points at 0.1 ml with Imject Alum Adjuvant (purchased from Thermofisher scientific, catalog number: 77161), that is, each mouse was injected with 25 ⁇ g of the immunogen.
  • TiterMax purchased from Sigma, catalog number: T2684
  • IP intraperitoneally
  • mice with high antibody titer in serum which tended to plateau were selected for splenocyte fusion.
  • Booster immunization was performed 3 days before splenocyte fusion, an antigen solution prepared with normal saline was injected subcutaneously and intraperitoneally (IP) at 50 ⁇ g/mouse.
  • ACK Lysing Buffer (purchased from Gibco, catalog number: A1049201) was added to lyse the red blood cells doped in the splenocytes to obtain a splenocyte suspension.
  • the cells were centrifugation at a rotating speed of 1500 r/min and washed with DMEM (purchased from Gibco, catalog number: 10566016) basal medium 3 times, and then mixed with mouse myeloma cells SP2/0 (purchased from ATCC, CRL-1581) at a ratio of 2 : 1 in terms of living cells.
  • DMEM purchased from Gibco, catalog number: 10566016
  • SP2/0 purchased from ATCC, CRL-1581
  • the fused cells were diluted into DMEM medium containing 20% fetal bovine serum (ExCell Bio, catalog number: FSD500) and 1 ⁇ HAT (purchased from Sigma, catalog number: H0262). The cells were added into a 96-well cell culture plate at 2 ⁇ 10 4 /200 ⁇ l/well, and cultured at 37°C and 5% CO 2 . After 14 days, ELISA was used to screen the supernatant of the fused cells, the positive clones were expanded into a 24-well plate for scale-up culture.
  • the medium was DMEM containing 10% (v/v) fetal bovine serum and 1 ⁇ HT (purchased from Sigma, catalog number: H0137), and the culture conditions were 37°C and 5% (v/v) CO 2 . After culturing for 3 days, the culture liquid of the scale-up culture in the 24-well plate was taken for centrifugation, and the supernatant was collected. The antibody subtype was analyzed for the supernatant, and ELISA and FACS were used to determine the binding activity to human CD19 protein and human CD19 positive cells (see examples 5.1-5.2 for the method for detecting binding activity).
  • the positive hybridoma cells in both the ELISA and FACS experiments were selected and subcloned using Medium D (purchased from STEMCELL, catalog number: 03810) in a 6-well plate. After about 7 days of subcloning, monoclones were selected and cultured in DMEM medium containing 10% FBS and 1 ⁇ HT for 2 days at 37°C and 5% CO 2 . ELISA was used for preliminary screening, and positive monoclones were selected and expanded into a 24-well plate for further culture.
  • ELISA and FACS were used to determine the antigen binding activity of the positive monoclones, and the optimal clone was selected and scale-up cultured in DMEM medium (purchased from Gibco) containing 10% (v/v) FBS at 37°C and 5% (v/v) CO 2 , and cryopreserved in liquid nitrogen, and thus the hybridoma cell of the present disclosure was obtained.
  • the hybridoma cells in the logarithmic growth phase were collected, and were fully lysed with Trizol (Invitrogen, Cat No. 15596-018) and stored at -80°C for testing.
  • Trizol Invitrogen, Cat No. 15596-018
  • GENEWIZ Suzhou was entrusted to determine the amino acid sequences of the variable regions of the light and heavy chains of the positive hybridoma clones, and 53 clones were obtained.
  • Biointron (Jiangsu) Biological Inc. was entrusted to clone the heavy chain variable region sequences of the aforementioned 53 clones into expression vector pcDNA3.4-B1HH1 containing a signal peptide and the heavy chain constant region of human antibody IgG1 (the sequence of the heavy chain constant region is as shown in SEQ ID NO: 433), respectively, clone the light chain variable region sequence of the clone in which the light chain is a Kappa chain into expression vector pcDNA3.4-B1HLK containing a signal peptide and the Kappa light chain constant region of human antibody IgG1 (the sequence of the light chain constant region is as shown in SEQ ID NO: 434), and clone the light chain variable region sequence of the clone in which the light chain is a Lambda chain into expression vector pcDNA3.4-BIHL5 containing a signal peptide and the Lambda light chain constant region of human antibody IgG1 (the sequence of the light
  • Human CD19-his protein (purchased from ACROBiosystems, catalog number: CD9-H52H2) was diluted with PBS to a final concentration of 4 ⁇ g/mL, and then added to a 96-well ELISA plate at 100 ⁇ l/well. The plate was sealed with a plastic film and incubated overnight at 4°C, the plate was washed 2 times with PBS the next day, and then a blocking solution [PBS+2% (w/w) BSA] was added for blocking at room temperature for 2 h. The blocking solution was poured off, and 100 nM gradiently diluted CD19 chimeric antibody or control antibody was added at 50 ⁇ l/well.
  • the binding results of the chimeric antibodies to human CD19 detected by ELISA are as shown in FIG. 5 and Tables 9-10. The results show that the chimeric antibodies all can bind to human CD19 at ELISA level.
  • the control antibody hIgG1 used in the identification or detection of CD19 chimeric antibodies in example 5, example 6, example 7 and example 8 was antibody anti-hel-hIgG1 (purchased from Biointron, catalog number: B11790 1) against hen egg lysozyme
  • the FMC63 antibody was FMC63-hIgG1 prepared in example 1.2
  • the 9G8 was 9G8-hIgG1 prepared in example 1.2.
  • the desired cells were scale-up cultured in a T-75 cell culture flask to the logarithmic growth phase.
  • the medium was aspirated, the cells were washed 2 times with a PBS buffer, and then digested with trypsin. After the digestion was terminated, the cells were washed 2 times with a PBS buffer; and for suspension cell Raji, the supernatant of the medium was discarded by directly centrifugation, and the cell pellet was washed 2 times with PBS.
  • the cell pellet was resuspended with [PBS + 2% (w/w) BSA] blocking solution to 2 ⁇ 10 6 cells/ml, and added to a 96-well FACS reaction plate at 50 ⁇ l/well, and then the sample to be tested was added at 50 ⁇ l/well, and incubated on ice for 2 h.
  • the obtained mixture was centrifuged and washed 3 times with a PBS buffer.
  • Alexa Fluor 647-labeled secondary antibody purchased from Jackson Immuno, catalog number: 109-605-088 was added at 50 ⁇ l/well, and the cells were incubated on ice for 1 h.
  • the obtained mixture was centrifuged and washed 5 times with a PBS buffer, and FACS (FACS CantoTM, purchased from BD Company) was used for detection and result analysis. Data analysis was performed by software (FlowJo) to obtain the mean fluorescence intensity (MFI) of the cells. And then software (GraphPad Prism8) was used for analysis, data fitting, and EC50 value calculation.
  • the analysis results are as shown in Table 11 and FIGs. 6-7 , indicating that the chimeric antibodies all can bind to human CD19 protein on the surface of Raji cells and CHO-K1-human CD19 cells.
  • an ELISA plate was coated with commercial murine CD19 (ACROBiosystems, catalog number: 50510-M08H) and monkey CD19 (ACROBiosystems, catalog number: 90051-C08H), respectively, and the ELISA detection was performed according to the method in example 5.1. None of the chimeric antibodies bound to murine CD19 protein at ELISA level.
  • the binding results of the chimeric antibodies to monkey CD19 detected by ELISA are as shown in FIG. 10 and Table 12, indicating that F5.11.2, F3.77.6, F3.118.14, F3.124.7, F3.148.10 have binding activity to monkey CD19 protein.
  • hIgG1 is negative control and NB151-89 is a positive serum capable of binding to monkey CD19-His protein.
  • HEK293T-monkey CD19 cells were subjected to FACS detection and data analysis according to the method in example 5.2.
  • the analysis results are as shown in Tables 13-14 and FIG. 11 , indicating that except for F3.23.10, F3.40.14, F3.74.4, F3.109.1, F3.113.16, F3.121.4, F3.148.10, F4.4.6, F4.33.12, F4.35.5, F4.37.6, F4.41.1, F4.58.6, F4.96.6, F4.145.9, F4.152.7, F4.180.1, F4.276.12, F5.7.1 and F6.5.20, all the other chimeric antibodies have binding activity to 293T cells overexpressing monkey CD19, and the EC50 shows that the highest binding activity is up to 0.73 nM.
  • the monkey peripheral blood mononuclear cells were extracted from fresh cynomolgus monkey peripheral blood (purchased from Shanghai Medicilon Inc.) according to the instructions of Ficoll-Paque Plus (purchased from GE Healthcae, catalog number: 171440-02). After the cell suspension was centrifuged, the cells were resuspended in PBS containing 1% BSA, and then the cells were counted. At the same time, murine antibody Brilliant Violet 605 anti-human CD20 (catalog number: 302334, purchased from Biolegend) having monkey CD20 cross-binding activity and the chimeric antibodies to be tested (1 nM, 10 nM and 100 nM) were added. The mixture was incubated for 1 h at room temperature.
  • APC-labeled secondary antibody anti-human IgG Fc (catalog number: 409306, purchased from Biolegend) was added. After incubation at room temperature in the dark for 30 minutes, the cells were washed 5 times, gently resuspended with PBS containing 1% BSA, and detected and analyzed by FACS (FACS CantoTM, purchased from BD Company), in which CD20 was used as a marker of B cells to gate the CD20-positive B cell population, the proportion of the chimeric antibody-labeled positive cells was analyzed, and the proportion of the chimeric antibody positive cell population to B cell population was calculated after treatments with the chimeric antibodies at the concentrations of 100 nM, 10 nM and 1 nM, respectively.
  • Anti-human CD19 antibodies were captured using Protein A chip (GE Helthcare; 29-127-558).
  • a sample buffer and a running buffer were HBS-EP+(10 mM HEPES, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P20) (GE Healthcare; BR-1006-69).
  • the flow-through cell was set to 25°C.
  • the sample block was set to 16°C. Both were pretreated with the running buffer.
  • an antibody to be detected was first captured with a Protein A chip, and then a single concentration of CD19 antigen protein was injected. The binding and dissociation processes of the antibody and the antigen protein were recorded, and finally Glycine pH 1.5 (GE Healthcare; BR-1003-54) was used to complete chip regeneration.
  • the binding was measured by injecting different concentrations of recombinant human CD19-His proteins in a solution for 240 s with a flow rate of 30 ⁇ l/min.
  • the concentration started from 200 nM (see the detailed results for the actual concentration in the test) and was diluted at 1 : 1, making a total of 5 concentrations.
  • the dissociation phase was monitored for up to 600 s and was triggered by switching from sample solution to running buffer.
  • CD19 protein contains 4 exons in the extracellular region, in which exons 1-3 are located at the membrane-distal end and exon 4 is located at the membrane-proximal end.
  • the antigen binding epitope of FMC63 is the membrane-proximal end (exon 4) of the extracellular region of CD19 protein.
  • human CD19 exon 1-3-His membrane-distal end was used to coat at 2 ⁇ g/mL according to the ELISA method in example 5.1. The results are as shown in FIG.
  • the antibodies can be divided into two categories: the first category does not bind to human CD19 exon 1-3-His, but has binding activity to human CD19-His, and the binding epitope is located at the non-membrane-distal end, such as F1-mab003; and the second category has binding activity to human CD19 exon 1-3-His, and has good binding activity to human CD19-His, and the binding epitope is located at the membrane-distal end, such as F3.23.10. Table 18.
  • heavy chain and light chain variable region germline genes having high homology to the murine antibody were selected as templates, respectively, and the CDRs of the murine antibody were grafted into the corresponding human template, to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the key amino acids in the framework sequence were back-mutated to the corresponding amino acids of the murine antibody to ensure the original affinity, and thus the humanized monoclonal antibody was obtained.
  • the CDR amino acid residues of the antibody were determined and annotated by the Kabat numbering system.
  • the humanization light chain templates of the murine antibody F3.121.4 were IGKV6-21*01/IGKV3-11*01 and IGKJ2*01, and the humanization heavy chain templates were IGHV1-3*01 and IGHJ6*01.
  • the CDRs of the murine antibody F3.121.4 were grafted into the human templates of the murine antibody respectively, and thus the corresponding humanized versions were obtained.
  • the key amino acids in the FR region sequence of the humanized antibody of F3.121.4 were back-mutated to the corresponding amino acids of the murine antibody to ensure the original affinity. If there are sites prone to chemical modification in the antibody, these sites are mutated to eliminate the risk of modification. See Table 19 for specific back mutation design.
  • Graft represents the grafting of the CDRs of the murine antibody into the FR region sequence of the human germline template
  • L46F represents the mutation of L at position 47 of Graft into F, and so on.
  • the numbering of back-mutated amino acids is according to the Kabat numbering.
  • variable region of the humanized antibody of F3.121.4 is as follows, where the underlined line represents a CDR region determined by the Kabat numbering system and the boxed character is a back mutation:
  • sequences of the humanization templates are as follows: FGQGTKLEIK (IGKJ2*01, SEQ ID NO: 445). WGQGTTVTVSS (IGHJ6*01, SEQ ID NO: 447).
  • Biointron (Jiangsu) Biological Inc. was entrusted to clone the heavy chain variable region sequence of the humanized antibody of F3.121.4 into expression vector pcDNA3.4-B1HH1 containing a signal peptide and the heavy chain constant region of murine antibody IgG1 (the sequence of the heavy chain constant region is as shown in SEQ ID NO: 448), and clone the light chain variable region sequence into expression vector pcDNA3.4-B1BLK containing a signal peptide and the light chain constant region of the murine antibody IgG1 (the sequence of the light chain constant region is as shown in SEQ ID NO: 449), and thus the expression vector of the humanized antibody of F3.121.4 was obtained.
  • the humanized antibody was prepared according to the method as shown in example 1.2.
  • the sequences of the heavy chain and light chain constant regions of the antibody are as follows:
  • ELISA detection and data analysis were performed according to the method in example 5.1 (the secondary antibody used was purchased from Jackson Immuno, catalog number: 115-035-003).
  • the analysis results are as shown in Table 21 and FIG. 15 , indicating that the humanized antibodies of F3.121.4 all can bind to human CD19 at ELISA level.
  • the control antibody mIgG1 used in the identification or detection of humanized antibodies in examples 10-13 was antibody anti-hel-mIgG1 (purchased from Biointron, catalog number: B118301) against hen egg lysozyme
  • the FMC63 antibody was FMC63-mIgG1 prepared in example 1.2
  • the 9G8 was 9G8-mIgG1 prepared in example 1.2.
  • FACS detection and data analysis were performed according to the method in example 5.2 (the secondary antibody used was purchased from Jackson Immuno, catalog number: 115-605-003).
  • the analysis results are as shown in Table 22 and FIGs. 16A-16D .
  • the results in Table 22 indicate that the humanized antibodies of F3.121.4 all can bind to human CD19 protein on the surface ofRaji cells and CHO-K1-human CD19 cells (2C8) (Table 22, and FIGs. 16A and 16C ).
  • the same method was used to detect the binding of the humanized antibodies of F3.121.4 to CHO-K1 cells and endogenous CD19-negative cells, MOLT-4 cells (purchased from ATCC, CRL-1582). The results are as shown in FIGs.
  • ELISA detection and data analysis were performed according to the method in example 6.1.
  • the analysis results show that the humanized antibodies of F3.121.4 do not bind to murine CD 19 protein and monkey CD 19 protein at ELISA level.
  • FACS detection and data analysis were performed according to the method in example 6.2.
  • the analysis results show that the humanized antibodies of F3.121.4 do not bind to 293T-monkey CD19 cells.
  • human CD19 exon 1-3-His membrane-distal end
  • Table 24 The results are as shown in Table 24, indicating that none of the humanized antibodies of F3.121.4 has binding activity to human CD19 exon 1-3-His protein. Therefore, it can be judged that the binding epitopes of the humanized antibody of F3.121.4 and the chimeric antibody F3.121.4 are both located at the non-membrane-distal end.

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